Tu1907 Curcumin Reduces the Inflammatory Factors of Reflux Esophagitis Through Non PKC Pathway in Part in Rat Model

Abstract

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. We have shown that NOX5-S was the major isoform of NADPH oxidase in FLO cells. NOX5-S mRNA expression is markedly increased in human Barrett's mucosal biopsies with dysplasia and in EA tissues, suggesting that NOX5-S may play an important role in this progression. In this study we examined the signal-transduction pathway of acid-induced NOX5-S expression in FLO EA cells. Acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of ROCK2 with ROCK2 siRNA and MEK1 with MEK1 siRNA, but not by knockdown of ROCK1 and MEK2. Conversely, overexpression of constitutively active ROCK2, but not constitutively active ROCK1, significantly enhanced NOX5-S expression and H2O2 production. In addition, acid treatment significantly increased the phosphorylation of MEK1 at threonine 292 (T292), but not at Serine 298 (S298). Overexpression of WT MEK1 and constitutively active MEK1 significantly increased NOX5-S promoter activity. Mutation of Threonine at 292 to Alanine abolished NOX5-S promoter activity and H2O2 production induced by overexpression of constitutively active MEK1. Acid-induced increase in ERK-2 phosphorylation was significantly reduced by Rho kinase inhibitor Y27632. Acidinduced increase in Rho kinase activity was not affected by knockdown of MEK1. In an in vitro kinase assay, we found that active ROCK2 increased MEK1 phosphorylation at T292. To test whether Rho kinases directly activate MEK1, we used synthesized peptides containing the flanking sequence of T292 and T292A in the ROCK2 kinase assay. We found that ROCK2 phosphorylated WT peptide DAAETPPRPRTPGRPLSSYGM, but did not phosphorylate T292A mutation peptide DAAETPPRPRAPGRPLSSYGM. Acid-induced increase in MEK1 T292 phosphorylation was significantly decreased by knockdown of ROCK2, but not of ROCK1. Overexpression of constitutively active ROCK2 remarkably increased MEK1 T292 phosphorylation, but not MEK1 S298 phosphorylation, in FLO EA cells. We conclude that acid-induced increase in NOX5-S expression and H2O2 production is mediated by sequential activation of ROCK2 and MEK1 in FLO EA cells. Activation of MEK1 depends on direct phosphorylation of MEK1 T292 by ROCK2. Supported by NIH NIDDK R01 DK080703.